The present invention relates to a PCR-based method of identifying various species of the genus Eimeria (commonly known as coccidia). More particularly, the present invention relates to a PCR-based method which is genus-specific, utilises novel PCR primers, and has the potential to identify species of Eimeria which may differ only by relatively minor sequence variations.
Bibliographic details of the publications referred to herein are collected at the end of the description.
Commonly used abbreviations in this text are: ITS, internal transcribed spacer; ITS-1, first internal transcribed spacer; ITS-2, second internal transcribed spacer; rDNA, ribosomal DNA; TBE, Tris-borate-EDTA; DPGE, denaturing polyacrylamide gel elctrophoresis; SSCP, single-strand conformation polymorphism.
Coccidiosis is a disease of animals and birds caused by protozoan parasites known as coccidia (Eimeria). This disease is of major economic importance for the poultry industry world-wide.
Eimeria species have complicated life cycles, details of which are well described. Briefly, when a sporulated (infective) coccidial oocyst is ingested, sporozoites are released to initiate asexual and sexual cycles that lead to the production of thousands of new oocysts, which are shed in the faeces of the host. These oocycts sporulate in the environment within days and then are infectious to naive birds. A single sporulated oocyst may give rise to thousands of progeny. Eimeria species produce lesions in the gut by destruction of the epithelial cells in which they develop and multiply, and cause trauma to the intestinal wall.
The clinical signs of coccidiosis include diarrhoea, which may be mucoid or bloody, and dehydration. These symptoms are generally followed by ruffled feathers, anaemia, listlessness, loss of weight, retraction of the head and neck and somnolence. Coccidiosis in laying hens is usually detected by a decrease in egg production. Infected growing birds, soon cease to grow satisfactorily. With highly virulent strains of Eimeria morality in chickens is generally very high.
Currently, seven species of Eimeria are recognised to infect chickens. These species differ considerably in their biology and pathogenicity (Mc Dougald et al, 1998). Being able to accurately identify Eimeria species and xe2x80x9cstrainsxe2x80x9d has major implications for diagnosis and control as well as for studying their epidemiology and population biology.
Traditionally, species of Eimeria have been identified by a variety of methods. For example, morphological features and/or morphometry of their oocysts or sporocysts (size, shape, length and width), their patterns of development, the nature of the lesions they produce, their predilection site(s) in the gut, sporulation times and reproductive index, or from the specific host from which they originate. However, these criteria can be unreliable (Eckert et al, 1995; Andrews and Chilton, 1999). Biochemical, immunological and molecular methods can overcome such limitations (Andrews and Chilton, 1999; Gasser, 1997) but may themselves have other limitations.
Polymerase chain reaction (PCR) methods of identifying species of Eimeria, using appropriate genetic markers, may be used as alternatives to the above mentioned traditional means, because of their ability to specifically amplify minute amounts of parasitic material (Stucki et al, 1993). However, such methods described to date are species-specific and may require the running of a number of different PCR reactions (using distinct pairs of species-specific oligonucleotide primers) in order to correctly identify a particular species of Eimeria.
For the molecular identification and classification of organisms analysis of critical specific genomic regions is required. One such region in eukaryotes is a part of the nuclear genome within the ribosomal DNA (rDNA) gene family. The rDNA of eukaryotes is a multigene family consisting of tandemly repeated units. Each unit comprises, an external transcribed spacer (ETS), the genes encoding the 18S, 5.8S and 28S rRNAs, separated by internal transcribed spacer regions (ITS-1 and ITS-2, respectively), and an intergenic spacer (IGS). Within this region the sequences of ITS-1 and ITS-2 provide reliable genetic markers for the identification of organisms to the species level because intraspecific variation in these sequences is usually low compared with higher levels of interspecific difference.
It has been demonstrated that ITS-1 and ITS-2 are useful genetic markers for the identification of species of Eimeria (Tsuji et al, 1997; Molloy et al, 1998; Schnitzler et al, 1999) or detection of population variation (Barta et al, 1998).
A PCR-based assay using species-specific primers in the ITS-1 for the typing of samples to species based on the detection of a product of a particular size on agarose gels has been developed (Schnitzler et al, 1999). However, such an assay does not allow sequence variation within a specific PCR product to be analysed and may further be disadvantaged by the fact that it is species-specific.
Further, Barta et al, 1998 have used a cloning/sequencing approach for the analysis of sequence variation both in the ITS-1 and ITS-2 sequences within E. maxima. Again, however, this approach may have limitations, especially where a large number of samples are to be analysed, as it is labour-intensive, time consuming and costly to perform. Moreover, it does not necessarily accurately define sequence variation among the different copies of rDNA, is species-specific, and can introduce artefacts into sequence data (Gasser, 1997).
Due to the economic impact of coccidiosis on the poultry industry, for example, it is important that species of Eimeria are readily identifiable, such that rapid diagnosis of disease and treatment may occur. Further, sensitive and reliable identification of Eimeria is desirable for the study of the epidemiology of the diseases and for controlling the purity of laboratory lines of Eimeria. Accordingly, there is a need to develop an assay for the rapid identification of species of Eimeria which does not have the limitations of previously described assays.
In one broad aspect of the present invention there is provided an oligonucleotide primer comprising at least 15 consecutive bases of the DNA sequence designated WW1 (SEQ ID NO: 31).
Preferably, the oligonucleotide primer comprises the sequence designated WW1 (SEQ ID NO: 31).
In a second broad aspect of the present invention there is provided an oligonucleotide primer comprising at least 15 consecutive bases of the DNA sequence designated WW3r (SEQ ID NO: 32).
Preferably, the oligonucleotide primer comprises the sequence designated WW3r (SEQ ID NO: 32).
In a third broad aspect of the present invention there is provided an oligonucleotide primer comprising at least 15 consecutive bases of the DNA sequence designated WW2 (SEQ ID NO: 33).
Preferably, the oligonucleotide primer comprises the sequence designated WW2 (SEQ ID NO: 33).
In a forth broad aspect of the present invention there is provided an oligonucleotide primer comprising at least 15 consecutive bases of the DNA sequence designated WW4r (SEQ ID NO: 34).
Preferably, the oligonucleotide primer comprises the sequence designated WW4r (SEQ ID NO: 34).
In a further broad aspect of the present invention there is provided a pair of PCR primers, one primer comprising at least 15 consecutive bases of the DNA sequence designated the WW1 (SEQ ID NO: 31) and a second primer comprising at least 15 consecutive bases of the DNA sequence designated WW3r (SEQ ID NO: 32).
Preferably, said pair of PCR primers comprises one primer comprising the DNA sequence designated WW1 (SEQ ID NO: 31) and a second primer comprising the DNA sequence designated WW3r (SEQ ID NO: 32).
In a further broad aspect of the present invention there is provided a pair of PCR primers, one primer comprising at least 15 consecutive bases of the DNA sequence designated WW2 (SEQ ID NO: 33) and a second primer comprising at least 15 consecutive bases of the DNA sequence designated WW4r (SEQ ID NO: 34).
Preferably, said pair of PCR primers comprises a primer comprising the DNA sequence designated WW2 (SEQ ID NO: 33) and a primer comprising the DNA sequence designated WW4r (SEQ ID NO: 34).
Preferably, said pairs of PCR primers as described herein are adapted to amplify specified regions of the rDNA of Eimeria in a genus-specific manner.
In a third broad aspect of the present invention there is provided a method of identifying Eimeria in a sample, said method comprising the steps:
providing a sample comprising genomic template DNA to be tested;
providing genomic DNA of one or more standards of known identity;
providing a pair of PCR primers selected from the group consisting of
(i) primers comprising at least 15 consecutive bases of the DNA sequence designated the WW1 (SEQ ID NO: 31) and WW3r (SEQ ID NO: 32); or
(ii) primers comprising at least 15 consecutive bases of the DNA sequences designated WW2 (SEQ ID NO: 33) and WW4r (SEQ ID NO: 34); and
amplifying by means of PCR a region of template DNA using said primer pair to produce one or more PCR products from said sample, and said one or more standard of known identity;
comparing said one or more PCR products derived from said sample against one or more PCR products of said one or more standard of known identity; and
identifying the species of Eimeria present within the sample.
Preferably, said pair of PCR primers comprises the primers comprising the sequences designated WW1 (SEQ ID NO: 31) and WW3r (SEQ ID NO: 32).
Alternatively, said pair of PCR primers comprises the primers comprising the sequences designated WW2 (SEQ ID NO: 33) and WW4r (SEQ ID NO: 34).
Preferably, two PCRs are run per sample to be tested, wherein each PCR uses a different primer pair.
Alternatively, one PCR is run per sample to be tested and both primer pairs are provided within said one PCR.
Preferably said one or more PCR products derived from said sample are compared against said one or more PCR products of said one or more standard of known identity by means of gel electrophoresis.
Preferably, the gel electrophoresis is DPGE.
More preferably, the gel electrophoresis is SSCP.
Alternatively, both DPGE and SSCP may be employed.
In a further aspect of the present invention there is provided the use of any one of the PCR primers as herein described for the identification of species of Eimeria.
The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of 2 or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.